Biological factors, such as coenzymes, cofactors, vitamins and others, play important roles in biological conversion processes. They are usually produced in small amounts in a whole cell (1×10−5–1×10−6). Determining the existence of, and thereafter isolating and purifying such biological factors from normal cells, however, is a difficult project.
Hyperimmunized eggs have been developed and have been shown to contain over-produced antibodies and, as set forth in U.S. Pat. No. 6,420,337, certain biological factors. The determination of exactly what biological factors exist in an egg is a difficult one that seems to be made easier with the hyperimmunization process. Hyperimmunization is performed by injection of polyvalent bacterial antigens into the target animal. It has been found that the amount of biological factors found in eggs from these hyperimmunized animals is increased, but by no more than one order of magnitude. Therefore, this small titer of biological factors in hyperimmunized egg makes locating and, thereafter, purification of biological factors difficult. As such, there is a need for an efficient process for recognizing, isolating, purifying or otherwise producing such biological factors.
Cytokines
The normal immune system is under a balance in which proinflammatory and anti-inflammatory cells and molecules are carefully regulated to promote normal host immune defense without the destruction of host's tissues. Once this careful regulatory balance is disturbed, nonspecific stimulation and activation can lead to increased amounts of potent destructive immunological and inflammatory molecules being produced and released. Thus, excess production of proinflammatory cytokines or production of cytokines in the wrong biological context, are associated with mortality and pathology in a wide range of diseases, such as malaria, sepsis, rheumatoid arthritis, inflammatory bowel disease, cancer and AIDS, among others.
Cytokines are pluripotent polypeptides that act in autocrine/paracrine fashions by binding to specific cellular receptors. Their secretion is important in determining the duration and intensity of an immune response. For example, in mice, distinct subsets of CD4+ T helper (Th) clones secrete what have classically been described as Th1 and Th2 cytokines. The cells produce interleukin-2 (IL-2) and interferon-γ (IFN-γ) and facilitate the cellular immune response. Th2 cells produce IL-4, IL-5, IL-6 and IL-10 and support the activation of immunoglobulin secreting cells. During the process of inflammation, cytokines such as IL-1β, IL-2, IL-6 and tumor necrosis factor-α (TNF-α) are released at the site of inflammation. These cytokines have pleiotropic effects and mediate a wide range of symptoms associated with inflammation.
A key cytokine, TNF-α, also known as cachectin, is a 17 kiloDalton protein composed of 157 amino acids and produced mainly by monocytes and activated macrophages. TNF-α has been shown to possess tumoricidal activity as well as a variety of physiological effects with most major organ systems. In the central nervous system, TNF-α is involved in fever, anorexia, and alterations in pituitary hormone release. In the cardiovascular system, TNF-α plays a role in shock, acute respiratory distress and capillary leakage syndrome (procoagulation). TNF-α is instrumental in the process of acute tubular necrosis and nephritis in the kidney and ischemia, colitis, and hepatic necrosis in the gastrointestinal system. It is also a key cytokine involved in the process of inflammation.
Inflammatory conditions are developed by multiple pathogenic processes, in which some key molecules are involved. For example, TNF-α, IL-1β, interlukin-2 (IL-2) and prostaglandin E2 (PGE2) are major contributors in developing inflammation. Inflammation can result in disease states, such as rheumatoid arthritis, septic arthritis, and juvenile osteoarthritis.
TNF-α is produced primarily by T-cells in response to inflammatory stimuli. TNF-α is a potent paracrine endocrine mediator of inflammatory and immune functions, and it modulates endothelial cell functions. For example, TNF-α can be detected in high concentrations in serum and synovial fluid from patients with active rheumatoid arthritis, and is suspected to have a primary role in the pathogenesis of rheumatoid arthritis.
IL-1β is involved in a wide variety of biological pathways, and is a remarkably potent molecule, able to induce its effects by triggering as few as one or two receptors per cell. As a signaling agent, IL-1β is effective at very low concentrations, even in the femtomolar range. IL-1β was first noted for inducing fever, augmenting lymphocyte responses, and stimulating the acute-phase response. The induction of an inflammatory reaction in response to infection is largely attributed to signaling by IL-1β.
Another T-cell-derived cytokine, IL-2, induces growth, differentiation, and functional activation in a variety of cells. For example, IL-2 promotes T-and B-cell growth and differentiation, immunoglobulin secretion by B-cells, NK cell growth and activity, and the production of other cytokines. IL-2 also plays an important function on the numerous inflammatory processes associated with immune cell growth and proliferation.
Prostaglandin E2 (PGE2)
Prostaglandin E2 (PGE2) is a “primary prostaglandin” found throughout many cells in the body. It is the predominate arachidonate metabolite and plays an important role in regulating vascular osmotic potentials as well as general conditions of the organism pertaining to body temperature, pain, immunosuppression, and inflammation. PGE2 is a major constituent in the inflammatory pathway of chronic inflammatory disease states.
A variety of enzymes are responsible for production of PGE2, and several of the same enzymes produce other prostaglandins, thromboxanes, and inflammatory mediators. For example, PGE2 is produced by both constitutively expressed and inducible forms of cyclooxygenase, COX-1, and COX-2, respectively. COX-2 is dramatically up-regulated when cells are exposed to certain mitogens (e.g. LPS) or cytokines (IL-1), providing a connection between cytokine signaling and PGE2 production.
Since TNF-α, IL-1β, IL-2, and PGE2 are the key inflammatory modulators, they play critical roles in the process of inflammation development.
Lipopolysaccharide (LPS) Analogs
A wide range of lipopolysaccharide (LPS) analogs interfere with cytokine expression in vitro and in vivo. For example, solid lipid nanoparticles (SLN) incubated with murine peritoneal macrophages cause a concentration-dependent decrease in IL-6 production. However, TNF-α and IL-12 are not suppressed. The synthetic lipid A analog SDZ MRL 953 protects against endotoxic shock and bacterial infection, as shown by a study in which 20 cancer patients were treated intravenously with escalating doses of SDZ MRL 953 followed by an intravenous application of endotoxin. Pretreatment with the lipid A analog markedly reduced the release of TNF-α, IL-1β, IL-8, IL-6, and G-CSF, suggesting that the pretreatment with SDZ MRL 953 in patients at risk may help to prevent complications of gram-negative sepsis.
LPS induced production of TNF-α can be inhibited in macrophages and in human monocytes and monoblastic U937 cells. A monosaccharidic lipid A analog, DY-9973, inhibits LPS-induced expression of TNF-α and IL-1β mRNA in U937 cells. In contrast, DY-9973 does not inhibit IL-1β-induced TNF-α production in U937 cells. Thus, monosaccharidic lipid A analog such as DY-9973 can inhibit LPS-induced activation of macrophages and reduce the lethal toxicity of LPS. Furthermore, an early endotoxin tolerance is induced by a nontoxic LPS derivative, monophosphoryl lipid A (MPL), against LPS infection.
In addition to lipid analogs, antibodies against LPS function as neutralizers to protect against LPS infection. Monoclonal antibody to lipid A suppresses the ability of lipid A and LPS from various gram-negative bacteria to induce TNF-α (36–67%) and IL-1 (30–98%) in murine peritoneal macrophages. This MAb also inhibited lipid A-induced TNF-α in mice (87%).
LPS analogs also upregulate cytokine expression. A different lipid A analog (DT-5461a) induces TNF-α, IL-1β, IL-6, and GM-CSF in murine macrophage. Furthermore, DT-5461a enhances production of various cytokines in cells through transcriptional enhancement.
Hyperimmunized Eggs
Various genera of the class Aves, such as chickens (gallus domesticus), turkeys, and ducks, produce antibodies in blood and eggs against immunogens that cause avian diseases, as well as against other immunogens. For example, LeBacq-Verheyden et al. (Immunology 27:683 (974)) and Leslie, G. A., et al. (J. Med. 130:1337 (1969)), have quantitatively analyzed immunoglobulins of the chicken. Polson et al. (Immunological Communications 9:495–514 (1980)) immunized hens against several proteins and natural mixtures of proteins, and detected IgY antibodies in the yolks of the eggs. Fertel et al. (Biochemical and Biophysical Research Communications 102:1028:1033 (1981)) immunized hens against prostaglandins and detected antibodies in the egg yolk. Jensenius et al. (Journal of Immunological Methods 46:63–68 (1981)) provide a method of isolating egg yolk IgG for use in immunodiagnostics. Poison et al. (Immunological Communications 9:475–493 (1980)) describe antibodies isolated from the yolk of hens that were immunized with a variety of plant viruses.
U.S. Pat. No. 4,357,272 discloses the isolation of antibodies from the yolks of eggs derived from hyperimmunized hens. The antibody response was elicited by repetitive injections of immunogens derived from plant viruses, human IgG, tetanus antitoxin, snake antivenins, and Serameba.
U.S. Pat. No. 4,550,019 discloses the isolation from egg yolks of antibodies raised in the hen by hyperimmunization with immunogens having a molecular or particle weight of at least 30,000. The immunogens used to hyperimmunize the chickens were selected from among plant viruses, human immunoglobulins, tetanus toxin, and snake venoms.
U.S. Pat. No. 4,748,018 discloses a method of passive immunization of a mammal that comprises parenterally administering purified antibody obtained from the eggs of an avian that has been immunized against the corresponding antigen, and wherein the mammal has acquired immunity to the eggs.
U.S. Pat. No. 5,772,999 discloses a method of preventing, countering or reducing chronic gastrointestinal disorders or Non-Steroidal Anti-Inflammatory Drug-induced (NSAID-induced) gastrointestinal damage in a subject by administering hyperimmunized egg and/or milk or fractions thereof to the subject.
U.S. Pat. No. 6,420,337 discloses a biological factor isolated from the egg of an avian hyperimmunized with an immunogenic mixture. The biological factor has been purified and sequenced and has been determined to activate certain pro-inflammatory cytokines, therefore referred to as a Cytokine Activating Factor.
European Patent Application No. 99967649.7 discloses an anti-inflammatory composition obtained also from the egg of an avian hyperimmunized with an immunogenic mixture. The anti-inflammatory composition has been partially purified from the egg and is shown to be effective in treating and preventing inflammation.
It has been reported that unsaturated fatty acids, such as omega-3 and -6 fatty acids (which can be found naturally in egg yolk), play an important role in the anti-inflammatory process. Recently, many investigators have been focused on the anti-inflammatory effects of omega-3 and -6 fatty acid enriched fish oil. These studies show that indeed omega fatty acids are effective in prevention of arthritis. The studies do not, however, set forth or present any evidence of the effect of omega fatty acids on pro-inflammatory cytokines or cytokines in general.
Some characteristics of omega fatty acids are that they are partially soluble in water, they have a maximum absorbance at 210–230 nm and their melting points are usually very low (−49° C. to 13.4° C.) due to their structure being that of a long-chain fatty acid.
It has not been reported in the literature that egg yolk phospholipids inhibit pro-inflammatory cytokines and/or synthesis of PGE2. Nor has it been reported that egg yolk contains any known lipopolysaccharide analogs (LPS analogs) that can function as competitors to LPS for the inhibition of pro-inflammatory cytokines and synthesis of PGE2.
The key to the present invention lies in the inventors' finding of a novel factor that occurs naturally in egg and has the ability to inhibit pro-inflammatory cytokines as well as synthesis of PGE2.